A binary probe-based DNAzyme cascade for rapid detection of MRSA/MSSA Project Summary This STTR Phase I project will develop a proof-of-concept molecular test for rapid detection of Staphylococcus aureus (SA) and differentiation between methicillin-resistant SA (MRSA) and methicillin-susceptible SA (MSSA) in the absence of PCR amplification. Binary DNAzyme probes will be designed to be complementary to an MRSA-specific gene or to an SA-specific gene. In presence of a target gene, the binary probes will be assembled into a catalytically active DNAzyme and activate a DNAzyme cascade, providing a visual or colorimetric readout. The Phase I research will focus on designing the binary DNAzyme probes, constructing the DNAzyme cascade, and demonstrating the technical feasibility for MRSA/MSSA screening. Compared to MSSA infections, MRSA infections have limited treatment options and result in significantly higher morbidity, mortality, and healthcare costs. Therefore, rapid and reliable detection of SA and differentiation between MRSA and MSSA will have significant clinical, economic, and societal impacts. If successfully developed, the proposed DNAzyme cascade approach will significantly improve the detection limit of current PCR-free molecular tests and have the potential to be developed into commercially viable, self-contained single-tube assays with minimum hands-on time and no requirement for sophisticated instrument. PUBLIC HEALTH RELEVANCE: Staphylococcus aureus, particularly MRSA, is a major cause of infections ranging from skin and soft tissue infections to invasive, life-threatening infections such as bacteremia, endocarditis, etc. The proposed PCR-free molecular test would address an urgent and unmet need for rapid, sensitive, and specific screening of MRSA and MSSA in an easy-to-use, cost- effective way, thereby allowing for timely and accurately treatment of MRSA/MSSA infections and helping reduce the associated healthcare costs.